I have recently expressed a protein in BL21 DE3 as a pET 20 (b+) construct (N-terminal His tag). The expression was quite good. I tried to purify the protein using HisLink Spin purification kit from Promega. I find that my eluate contains most of the host proteins and very little quantity of my protein. tried increasing the NaCl conc in te wash buffers (500 mM) and I even tried adding more imidazole to the wash buffer (upto 20 mM). I also tried adding more imidazole to the elution buffer (upto 750 mM)> Even then I keep getting a lot of other protein bands and very little or none at all of my protein of interest. My question is if my protein has not been transcribed witht he His tag, it should be seen in the lysate flow through or the wash flow throughs or even in the final elution witht he other proteins. Then y cannot I detect it? The kit calls for 700 ul of bacterial culture as the start volume (While running the SD-PAGE I also run a lane containing protein from 700 ul culture - lysed in loading dye and of which 20 ul is loaded on the gel; for comparison purpose).

Can someone tell me whats happening or any suggestions at all??? Thanks in advance.

-smartsunny-

QUOTE (smartsunny @ Apr 7 2008, 04:55 AM)

Hello everybody,

I have recently expressed a protein in BL21 DE3 as a pET 20 (b+) construct (N-terminal His tag). The expression was quite good. I tried to purify the protein using HisLink Spin purification kit from Promega. I find that my eluate contains most of the host proteins and very little quantity of my protein. tried increasing the NaCl conc in te wash buffers (500 mM) and I even tried adding more imidazole to the wash buffer (upto 20 mM). I also tried adding more imidazole to the elution buffer (upto 750 mM)> Even then I keep getting a lot of other protein bands and very little or none at all of my protein of interest. My question is if my protein has not been transcribed witht he His tag, it should be seen in the lysate flow through or the wash flow throughs or even in the final elution witht he other proteins. Then y cannot I detect it? The kit calls for 700 ul of bacterial culture as the start volume (While running the SD-PAGE I also run a lane containing protein from 700 ul culture - lysed in loading dye and of which 20 ul is loaded on the gel; for comparison purpose).

Can someone tell me whats happening or any suggestions at all??? Thanks in advance.

Throw away the purification kit. But some Ni-NTA resin from Qiagen. Grow 1L BL21, induce 600mM IPTG for 2hours at 25C, spin down and lyse the cells in 50mM buffer and 400mM NaCL + protease inhibitors and DNAse. Spin for 15K 30min, Add 100uL Ni-NTA bead to supernatant, bind for 1 hour. spin down beads, discard flowthrough and wash with 2mL 20mM Imidazole then 2mL 30mM Imidazole. Elute in 200ul of 100, 200, and 300 Imidazole. Run elutions on gel. Good Luck